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determine its association with aggressive disease charac-

teristics. Better understanding of the relevance of this

tumor marker in this population has possible implica-

tions for adjuncts in diagnosis, treatment planning, and

targeted therapy.

MATERIALS AND METHODS

Patients 0 to 18 years old were selected for a retrospective

chart review if they underwent surgery for PTC at our institu-

tion (Primary Children’s Medical Center, The University of

Utah Hospital, Huntsman Cancer Hospital) between 1999 and

2012. Institutional review board approval was obtained (The

University of Utah IRB 00057453).

A retrospective chart review was performed using institu-

tional electronic medical records. Patient demographic factors

and disease characteristics (tumor size, lymphatic/distant

metastases, surgical and adjuvant treatment rendered, lympho-

vascular invasion, extrathyroidal extension, recurrence, histol-

ogy) were obtained and kept in a secure patient database.

Metastases, age at diagnosis, completeness of resection, inva-

sion, and size of the tumor (MACIS) score was calculated.

Tumor samples for the study subjects were obtained from

archived pathologic specimens. Formalin-fixed paraffin-embed-

ded (FFPE) tissue blocks were used to prepare hematoxylin and

eosin slides to identify areas of tumor cells. Aniline blue-stained

slides were processed from adjacent slices of FFPE tissue, and

microdissection of tumor cells was performed. A single patholo-

gist (

.) performed all tumor microdissection. DNA was then

extracted using a standardized technique.

Exon 15 of the BRAF gene was amplified using polymerase

chain reaction with primers as shown in Figure 1. After the ampli-

fication, mutation status was determined by pyrosequencing using

the Qiagen PyroMark Q24 pyrosequencer (Qiagen, Venlo, the

Netherlands) following the manufacturer’s instructions, as has

been outlined previously.

Sequence analysis was performed

using the Pyromark Q24 version 1.0.10 software in the allele

quantification (AQ) analysis mode, using pyrograms as shown in

Figure 2. The assay operates with a sensitivity of 5% of alleles.

Statistical analysis was performed with SPSS software

(IBM, Armonk, New York). Fischer exact test was used to mea-

sure the association of the BRAF V600E mutation between

binary variables (lateral and central neck metastases, pulmo-

nary metastases, histology, lymphovascular invasion, extrathyr-

oidal extension, recurrence). A two-tailed

test was used to

measure association between the BRAF mutation and continu-

ous data (tumor size, age, MACIS score).

A review of the literature was performed by searching

PubMed for “papillary thyroid carcinoma” and “BRAF” with lim-

its applied for patients aged 0 to 18 years, as well as additional

text search strings for “children” or “pediatric” or “adolescent.”

Results of these relevant studies were summarized.

RESULTS

Archived tumor specimens were available for 19 of

27 pediatric patients who initially fit inclusion criteria.

Demographic data are shown in Table I. Ages ranged

from 2.8 to 18 years (median, 13.6 years). Two patients

had previously undergone thyroidectomy, whereas the

remainder had thyroidectomy performed at our facility.

Average tumor size was 2.18 cm (range, 3 mm to 4.2

cm). Five patients had papillary microcarcinoma,

whereas the remainder had tumors

1 cm. The average

MACIS score was 5.1. Thirteen patients underwent cen-

tral compartment neck dissection, nine underwent lat-

eral neck dissection, including two who underwent

bilateral neck dissections. Thirteen patients (68.4%) had

metastases to the central neck, eight (42.1%) had lateral

neck metastases, and five (26.3%) had pulmonary metas-

tases. Two patients experienced regional recurrence. The

BRAF V600E mutation was present in seven patients

(36.8%). Eleven patients had classic PTC (including one

with partial tall cell morphology), seven had a follicular

variant of PTC, and one had an oncocytic variant. Seven

of the 11 (63.6%) samples with classical PTC were BRAF

V600E positive. All samples with variant pathology

showed wild-type BRAF.

PTC histology was significantly associated with the

presence of the BRAF V600E mutation (

.013,

Cramer’s effect size V

0.651). Similarly, FVPTC histol-

ogy was negatively associated with the BRAF V600E

mutation (

.017). There was no association of the fol-

lowing variables with wild type or BRAF V600E (Table

II): presence of lateral neck metastases (50.0% vs.

28.5%,

.633), central neck metastases (75.0% vs.

57.1%,

.617), pulmonary metastases (42% vs. 0%,

.106), average tumor size (2.23 cm vs. 2.08 cm,

0.176,

.863), average age (12.9 years vs. 14.8

years,

1.221,

.239), lymphovascular invasion

(77.8% vs. 60.0%,

.580), extrathyroidal extension

(62.5% vs. 60%,

1.00), and incidence of papillary

microcarcinoma (36.4% vs. 16.7%,

.851). MACIS

score approached significance (5.59 vs. 4.23,

.087)

Fig. 1. Primers used the polymerase chain reaction to amplify

exon 15 of the

BRAF

gene.

Fig. 2. Sequence analysis using the Pyromark Q24 version 1.0.10

software in the allele quantification analysis mode using

pyrograms.

Givens et al.: BRAF V600E and Pediatric Thyroid Carcinoma

Laryngoscope

124:

September

2014

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